The first major accomplishment of this project was publication of the results of the second trial of rhIL-7 in humans (Sportes et al, J of Exp Med, 2008;205;1701) . This trial incorporated extensive biologic studies that provided insight into the basis for the dramatic changes in T cell homeostasis induced by IL-7. We demonstrated that IL-7 causes a broad repertoire of T cells to undergo cell cycling and to resist programmed cell death, resulting in rapid, significant expansion of both blood and tissue T cells. The expansion was tightly regulated by dynamic regulation of the IL-7 receptor, thus preventing dangerous increases in lymphocytes with IL7 therapy. Because IL-7 selectively expands the youngest or most naive T cell populations, T cells in IL-7 treated patients resembled T cells normally present at young ages. Thus, IL-7 has an anti-aging effect on the immune system. This capacity to expand a broad pool of nave cells is unique among cytokines currently under study and is predicted to be very important for immune competence, especially in the context of T cell depletion. We saw little to no evidence of toxicity with IL-7, providing a solid basis for Phase II trials of this agent in combination with directed immunotherapy and for studies of IL-7 in HIV infection. A second major accomplishment in this project during FY2009 was the development of a new regimen that can support adoptive immunotherapy without inducing lymphodepletion in patients. Many groups are currently subjecting patients to lymphocyte depletion as a means for improving the milieu within which adoptively transferred cells may proliferate and survive in vivo. This approach brings with it toxicities associated with the regimens used to induce lymphopenia, and the long term morbidity or chronic immunosuppression. This work also demonstrated that lymphopenia has substantial limitations in terms of its ability to support adoptive immunotherapy that are related to a limited immune repertoire for epitope spreading, diminished functionality of T cells in the lymphopenic milieu as well as diminished immune surveillance. This work demonstrated that provision of increased IL-7 and depletion of regulatory T cells would provide an equivalent or superior milieu for adoptive immunotherapy. We utilized the B16 melanoma model and adoptively transferred T cells with specificity for the immunodominant antigen on this tumor. We observed that adoptive immunotherapy administered to lymphopenic mice provided only a modest benefit compared to T cell replete mice however specific depletion of CD25+ T cells using a monoclonal antibody substantially improved the effectiveness of adoptive immunotherapy. Such mice showed substantially improved survival following adoptive immunotherapy compared to lymphopenic mice. We conclude that lymphopenia should not be considered a requisite setting for adoptive immunotherapy and that targeted approaches that provide the specific components of the altered physiology of lymphopenia are preferable and will be more effective in the long run. This work was accepted for publication in July 2009 (Cui et al, Blood, 2009). A third major accomplishment of this project during FY2008 involved the demonstration that our clinically qualified master cell bank of artificial antigen presenting cells effectively expand cytolytic NK cells and can therefore serve as a critical reagent for developing immune based therapies for cancer. We had previously demonstrated that these cells can effectively expand cytolytic CD8+ T cells and this work extends that to NK cells. NK cells expanded with this reagent effectiveless lyse tumors of multiple histologies including essentially all pediatric sarcomas tested (rhabdomyosarcoma, osteosarcoma, Ewings sarcoma, etc.) as well as acute lymphoblastic leukemia cells, both primary cells and tumor cell lines. We are currently completing submission of an IND and a clinical trial to investigate the use of aAPC activated NK cells in the context of non-myeloablative hematopoietic stem cell transplantation. A fourth major accomplishment was the demonstration that interferon gamma signaling on antigen presenting cells prevents the development of graft-versus-host disease and enhance the capacity for tumor vaccines to eradicate disease in this setting. This is a novel axis which has not previously been implicated in GVHD and this model provides the first serious assessment of the relative impact of the allogeneic milieu in modulating vaccine based responses. This work was published in Blood (Capitini et al, Blood, 2009). During the past year we also collaborated on a project with Dr. Christoph Raders laboratory to investigate the biology of an anti-NKG2D monoclonal antibody, which can be used to either block or activate NK mediated effects (Kwong et al, J Mol Bio 2008). Using our expertise in NKG2D biology based upon studies of NKG2D expression on CD8+ T cells in lytic activity of Ewings sarcoma cells, we were able to demonstrate that the anti-NKG2D monoclonal developed by the Rader lab was bound specifically to NKG2D and that the Fab fragment could be used to inhibit NKG2D binding to its ligands. Importantly, when crosslinked, this same molecule was also effective at enhancing NKG2D mediated killing, a feature that is potentially exploitable for clinical applications. We also published a manuscript demonstrating that tumor apoptotic bodies and tumor lysates are superior to peptides in generating antitumor immunity, a result which directly impacts our current and future approaches to developing immune based therapies for patients with cancer (Fry et al, Cancer Imm and Immunoth, 2009). Finally, we hosted an international meeting of essentially all of the scientific groups involved in developing immune based therapies for childhood cancer. This resulted in several collaborations and publication of a summary manuscript (Capitini et al, J of Ped Hem/Onc, 2009).